This invention relates to an apparatus for efficient preparation of the crystals of biomacromolecules such as proteins and nucleic acids. The invention also relates to a method for preparing the crystals of biomacromolecules in which the concentration in solution of a biomacromolecule such as a protein or nucleic acid and the concentration of a precipitant are adjusted on a real-time basis to construct a crystal phase diagram for the biomacromolecule and, in addition, on the basis of the constructed crystal phase diagram, the solution of the biomacromolecule is shifted between a supersaturated and a metastable region to effect nucleation and growth of the biomacromolecule's crystal.
Following the success in mapping of the human genome, an international project has launched with the hope of determining the three-dimensional structures (about 10,000 in number) of the structural families of all proteins in five years. With a view to determining 3,000 out of those 10,000 proteins primarily by x-ray analysis and NMR, Japan has started the so-called Project 3000 Proteins. It is not clear how many of the 3,000 proteins are going to be determined by x-ray analysis of crystal structures but 2,000 would be a minimum number.
In the x-ray analysis of crystal structures, a single crystal to be subjected to structural analysis is essential and preparing such a single crystal has been a bottleneck in the art.
Vapor diffusion is one of the conventional methods commonly employed to prepare single crystals. In this method, the moisture in a solution that lies in the unsaturated region of a phase diagram for the molecule to be crystallized is vaporized, whereby the concentration of the molecule to be crystallized and that of a precipitant are increased so that the state of the solution is shifted to the supersaturated region in the phase diagram, thereby starting the growth of a crystal and causing it to grow. In this method of crystal growth, the concentration of the molecule to be crystallized and that of the precipitant are not controlled artificially, so in order to prepare a single crystal of a certain molecule to be subjected to structural analysis, it has been necessary to prepare solutions of the molecule under a huge number of conditions and cause it to crystallize out of the individual solutions.
In addition, as the solvent vaporizes, the concentration of the molecule to be crystallized and that of the precipitant will only change to increase, so once the solution is shifted into the unsaturated region with a view to forming crystal nuclei, it is difficult to readjust the conditions of the solution to enter the metastable region which provides more preferred conditions for crystal growth.
On account of these problems, single crystal preparation by the vapor diffusion method has been considerably wasteful both time-wise and money-wise.
In order to solve the above-mentioned problems with the vapor diffusion method, a robot-assisted method for high-throughput crystal growth has been devised (WO 01/92293; Wilding, P. and Kricka, L. J., TIBTECH, 17, 465-468 (1999)). However, this is no more than a proposal for handling protein solutions by a machine, rather than humans, in the vapor diffusion method and it by no means provides a new technique for crystal growth. As a matter of fact, the problems with the vapor diffusion method are yet to be solved by the proposal.
Even today, the essence of structural biology lies in unraveling the physiological functions of a certain molecule at the atomic level by determining the hydrogen atoms and structure of hydration through high-resolution structural analysis and neutron diffractometry, both being intended to attain a resolution in the neighborhood of 1 angstrom. To meet this need, single crystals are required that are either of good quality or larger than 1 mm3.
Single crystals that satisfy these requirements are difficult to obtain by just repeating the conventional mechanical trial-and-error approach and it is strongly desired to develop a method and an apparatus that facilitate the production of biomacromolecules as single crystals having the required level of quality.